(A) Resolution of two different RNAPII complexes by Sepharose CL-4B gel filtration chromatography. The upper panel summarizes the Western blot results shown below. The lower panel shows Western blots of fractions of the Sepharose CL-4B gel filtration column. Blots were probed with antibodies as indicated at the side of each blot. IIF (RAP74), the RAP74 subunit of TFIIF cycC, cyclin C. Input (1%; IN) and fraction numbers are indicated on the top of the lower panel. The last row shows the transcription activity of the column fractions obtained by using the adenovirus major late promoter in a system reconstituted with TBP, TFIIB, and TFIIH. (B) The amount of acetate incorporated into core histones and the protein concentration of each Mono S (see Materials and Methods) column fraction. (C) Coelution of CBP, PCAF, RNAPII (RPB1), BAF47, cyclin C, and CDK8 by Mono S ion exchange chromatography. Fractions from a similar Mono S column were analyzed by Western blotting using antibodies against CBP, PCAF, RPB1, BAF47, cyclin C, and CDK8.

(A) Western blot analysis of the immunoprecipitates using antibodies against CDK8 and hSrb7. Immunoprecipitates (IP) of anti-CDK8 (lane 2), anti-Srb7 (lane 3), and control (lanes 4 and 5) antibodies and 10% of the input protein fraction (lane 1) were analyzed by SDS–5 to 12% PAGE. The Western blots were probed for CBP/p300 and RNAPII (top panel), PCAF (middle panel), and Swi-Snf component BAF47 (bottom panel). (B) Western blot analysis of the immunoprecipitates using antibodies against histone acetyltransferases PCAF and CBP. Immunoprecipitates of anti-CTD (lane 2), anti-PCAF (lane 3), anti-CBP (lane 4), and control (lane 5) antibodies and 10% of the input protein fraction (lane 1) were analyzed by SDS–5 to 12% PAGE and Western blot analysis. The Western blots were probed for CBP/p300 (top panel), PCAF (second panel), RNAPII (third panel), and Swi-Snf component BAF47 (bottom panel).

(A) Western blot analysis of the fractions derived from phosphocellulose and DEAE-cellulose columns. Western blots of eluates of 0.1 (0.18 mg of protein per ml), 0.3 (0.12 mg of protein per ml), 0.5 (0.08 mg of protein per ml), and 1.0 M KCl (0.16 mg of protein per ml) from a phosphocellulose column (left panel) and 0.1 (0.03 mg of protein per ml) and 0.35 M KCl (0.18 mg of protein per ml) eluates of the DEAE-cellulose column (right panel) derived from the phosphocellulose 0.5 M KCl fraction are shown (see Materials and Methods). Each eluate (20 μl) was loaded on an SDS–5 to 20% polyacrylamide gel for Western blot analysis. Each blot was analyzed for CDK8, cyclin C (cyc C), hSrb7, BAF190, BAF47, the largest subunit of RNAPII (RPB1), the 89-kDa subunit of TFIIH (ERCC3) (TFIIH p89), the large subunit of TFIIF (RAP74), the small subunit of TFIIE (p34), TFIIB, and the histone acetyltransferases CBP and PCAF. (B) Affinity-purified CDK8 complex contains hSrb proteins, Swi-Snf subunits (BAFs), and substoichiometric amounts of RNAPII but not GTFs, the corepressor DRAP1, or the coactivator PC4. Western blots of input (I), flowthrough (FT), and eluate (Elute) of anti-CDK8 (CDK8 col) and control antibody (con. col) columns are shown. Blots were probed for BAF190, BAF170, BAF155, BAF60, BAF47, CDK8, cyclin C, hSrb7, RPB1, TFIIF (RAP74), TFIIH (p62), TFIIE (p56), TBP, TFIIB, DRAP1, and PC4. (C) A silver-stained SDS-polyacrylamide gel containing the affinity-purified CDK8 complex and the Swi-Snf complex shows multiple common polypeptides. A silver-stained gel containing affinity-purified CDK8 complex (left) and Swi-Snf complex (right) was aligned to show common polypeptides (>29 kDa). Another silver-stained gel containing bands from 29 to 10 kDa showing polypeptides derived from the affinity-purified CDK8 complex is at the bottom of the left lane. Polypeptides were identified by Western blot analysis as indicated. Other polypeptides in this fraction which are common to the polypeptides present in the DEAE-cellulose flowthrough fraction-derived affinity-purified CDK8 complex were identified by microsequencing (67a).

(A) Comparison of Western blots of affinity-purified CDK8 complexes isolated from the DEAE-cellulose flow-through (Elute DE FT) and DEAE-cellulose-bound (Elute DE Bound) fractions. (B) Heparin or ethidium bromide does not disrupt the interaction of the components of the complex. Western blots of CDK8 complex affinity purified in the absence (second lane from left) or the presence of ethidium bromide (lane + EtBr), heparin (lane + Heparin), and Sarkosyl (lane + Sarkosyl) and input (lane 1; 1%) are shown. Either ethidium bromide (400 μg/ml), heparin (16 μg/ml), or sarkosyl (0.1%) was added as indicated to the DEAE-cellulose-bound fraction during the incubation with the antibody column as well as during the washes. (C) The affinity-purified CDK8 complex alters the linking number of reconstituted plasmid chromatin. Buffer (lane 1), highly purified Swi-Snf (Superose 6 column fraction; lane 2), the control antibody fraction (lane 3), and the affinity-purified CDK8 complex (lane 4) were incubated with plasmid reconstituted into chromatin in vitro (see Materials and Methods). Plasmid DNA was extracted and analyzed for a change in linking number.

(A) Silver-stained gel of core RNAPII containing the phosphorylated (IIo) and nonphosphorylated (IIa) forms of the large subunit that was used in the interaction experiments shown in panels C and D. (B) Western blot analysis reveals the absence of RHA in the RNAPII preparation used in the interaction experiments. Lane 1 shows a Western blot of a crude fraction containing RHA, and lane 2 shows a Western blot of the RNAPII (50% of input) preparation used in the interaction studies. Blots were analyzed for RHA and subsequently reprobed for the largest subunit of RNAPII (IIa and IIo). (C) PCAF directly interacts with the phosphorylated form of RNAPII. Different deletion mutant polypeptides of PCAF (77) were immobilized on beads through their N-terminal FLAG tags and were incubated with a mixture of phosphorylated and nonphosphorylated forms of RNAPII (I; 10% of input). Immunoprecipitates (IP) were extensively washed as described in Materials and Methods and were analyzed by Western blotting using anti-CTD antibodies (top gel) and anti-FLAG antibodies (bottom gel). The diagram above the blots shows the different deletion PCAF proteins that were used in the assay. WT, wild type. (D) p300 directly interacts with the nonphosphorylated form of RNAPII through its C-terminal domain. Experiments were performed as described for panel C. The diagram at the top illustrates the different truncated p300 proteins (53) used. The top blot shows the results of Western blotting with anti-CTD and anti-FLAG antibodies; the bottom blot shows the results of Western blotting with anti-FLAG antibodies. IgG, immunoglobulin G.